Functional ligands to drug compounds

ABSTRACT

The present invention relates functional ligands to target molecules, particularly to functional nucleic acids and modifications thereof, and to methods for simultaneously generating, for example, numerous different functional biomolecules, particularly to methods for generating numerous different functional nucleic acids against multiple target molecules simultaneously. The present invention further relates to functional ligands which bind with affinity to target molecules, such as drug compounds, such as levamisole and cocaine.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a 371 national stage application of PatentCooperation Treaty International Application Ser No. PCT/US18/20319,which claims the benefit and priority of the following U.S. provisionalpatent applications: Ser. No. 62/464,477, filed Feb. 28, 2017, entitled,“FUNCTIONAL LIGANDS TO LEVAMISOLE”, Ser. No. 62/501,738, filed May 5,2017, entitled, “FUNCTIONAL LIGANDS TO DRUG COMPOUNDS”, and Ser. No.62/574,125, filed Oct. 18, 2017, entitled “FUNCTIONAL LIGANDS TOLEVAMISOLE”; the contents all of which are hereby incorporated byreference in their entireties. This application is a 371 national stageapplication of Patent Cooperation Treaty International ApplicationPCT/US18/19285, which claims the benefit and priority of the followingU.S. provisional patent applications: Ser. No. 62/462,311, filed Feb.22, 2017, entitled, “FUNCTIONAL LIGANDS TO CORTISOL”, Ser. No.62/512,676, filed May 30, 2017, entitled, “FUNCTIONAL LIGANDS TOCORTISOL”, and Ser. No. 62/464,287, filed Feb. 27, 2017, entitled“FUNCTIONAL LIGANDS TO OPIOIDS”; the contents all of which are herebyincorporated by reference in their entireties.

FIELD OF THE INVENTION

This invention relates to functional ligands to target molecules,particularly to functional nucleic acids and modifications thereof, moreparticularly to functional ligands with binding affinity to targetmolecules such as drug compounds, such as levamisole and cocaine.

SEQUENCE LISTING

Deoxyribonucleic acid (DNA) sequences, which are disclosed in the ASCIItext file entitled “PLEVAPC01_ST25.txt”, created on Feb. 28, 2018 and of72.6 KB in size, which is incorporated by reference in its entirety,herein are intended to include other aptamers incorporatingmodifications, truncations, incorporations into larger molecules orcomplexes, and/or other aptamers having substantial structural orsequence homology, for example, greater than 75% sequence homology, aswell as RNA and/or other non-DNA/RNA aptamers. The disclosed aptamersmay also bind to homologous proteins or molecules from organisms otherthan the organisms listed herein, to recombinant or non-recombinantversions of the proteins or molecules, to modified versions of theproteins or molecules, to proteins or molecules from sources other thanthe source listed herein. The aptamers are artificial, non-naturallyoccurring sequences designed and/or selected for specific and/or highaffinity binding to a target molecule. Non-naturally occurring sequencesof aptamers, may also not be present in naturally occurring systems orsituations, such as by, for example, not being already present or havinga pre-existing function in a naturally occurring setting. The indicationof the species and source of the target proteins or molecules is givenfor reference only and is not intended to be limiting. Withoutlimitation as to target molecules to which they may bind and withoutbeing bound to any particular theory, SEQ IDs 1-190 may generally bindto the target molecule levamisole, and SEQ IDs 50, 57, 191-290 maygenerally bind to the target molecule cocaine. SEQ ID 291 may generallybe utilized as a leading or priming sequence appended to the 5′-end ofany of the aptamer sequences, and SEQ ID 292 may generally be utilizedas a trailing or priming sequence appended to the 3′-end of any of theaptamer sequences, or vice versa or any combination thereof.

BACKGROUND OF THE INVENTION

Aptamers, which are nucleic acid ligands capable of binding to moleculartargets, have recently attracted increased attention for their potentialapplication in many areas of biology and biotechnology. They may be usedas sensors, therapeutic tools, to regulate cellular processes, as wellas to guide drugs to their specific cellular target(s). Contrary to theactual genetic material, their specificity and characteristics are notdirectly determined by their primary sequence, but instead by theirsecondary and/or tertiary structure. Aptamers have been recentlyinvestigated as immobilized capture elements in a microarray format.Others have recently selected aptamers against whole cells and complexbiological mixtures. Aptamers are typically characterized by binding totheir target molecules via non-Watson-Crick (i.e. non-hybridization)mechanisms, such as by intermolecular forces resulting from thesecondary or tertiary structure of the aptamer. This is especially trueof non-nucleic acid target molecules where Watson-Crick mechanismstypically do not apply.

Aptamers are commonly identified by an in vitro method of selectionsometimes referred to as Systematic Evolution of Ligands by EXponentialenrichment or “SELEX”. SELEX typically begins with a very large pool ofrandomized polynucleotides which is generally narrowed to one aptamerligand per molecular target. Once multiple rounds (typically 10-15) ofSELEX are completed, the nucleic acid sequences are identified byconventional cloning and sequencing. Aptamers have most famously beendeveloped as ligands to important proteins, rivaling antibodies in bothaffinity and specificity, and the first aptamer-based therapeutics arenow emerging. More recently, however, aptamers have been also developedto bind small organic molecules and cellular toxins, viruses, and eventargets as small as heavy metal ions.

Cocaine (Methyl (1R,2R,3S,5S)-3-(benzoyloxy)-8-methyl-8-azabicyclo[3.2.1]octane-2-carboxylate), also known as coke, is a strong stimulantmostly used as a recreational drug. Cocaine and its major metabolitesmay be quantified in blood, plasma, or urine to monitor for abuse,confirm a diagnosis of poisoning, or assist in the forensicinvestigation of a traffic or other criminal violation or a suddendeath. Cocaine may be present in various salt forms as well, typicallyas a hydrochloride salt.

Levamisole ((S)-6-Phenyl-2,3,5,6-tetrahydroimidazo[2,1-b][1,3]thiazole),sold under the trade name Ergamisol among others, is a medication usedto treat parasitic worm infections. Specifically it is used forascariasis and hookworm infections. Levamisole has increasingly beenused as a cutting agent in cocaine sold around the globe with thehighest incidence being in the US. Levamisole adds bulk and weight topowdered cocaine (whereas other adulterants produce smaller “rocks” ofcocaine) and makes the drug appear purer. Other rationales forlevamisole's rise as an adulterant include possible stimulant effects, asimilar appearance to cocaine, and an ability to pass street puritytests. Levamisole suppresses the production of white blood cells,resulting in neutropenia and agranulocytosis. With the increasing use oflevamisole as an adulterant, a number of these complications have beenreported among cocaine users. Levamisole has also been linked to a riskof vasculitis and two cases of vasculitic skin necrosis have beenreported in users of cocaine adulterated with levamisole. Levamisole maybe quantified in blood, plasma, or urine as a diagnostic tool inclinical poisoning situations or to aid in the medicolegal investigationof suspicious deaths involving adulterated street drugs.

SUMMARY OF THE INVENTION

The present invention relates functional ligands to target molecules,particularly to functional nucleic acids and modifications thereof, andto methods for simultaneously generating, for example, numerousdifferent functional biomolecules, particularly to methods forgenerating numerous different functional nucleic acids against multipletarget molecules simultaneously. The present invention further relatesto functional ligands which bind with affinity to target molecules, moreparticularly to functional ligands with binding affinity to targetmolecules such as drug compounds, such as levamisole and cocaine. Thepresent invention further relates to methods for generating, forexample, functional biomolecules, particularly to functional nucleicacids, that bind with functional activity to another biomolecule, suchas a receptor molecule. More than one or multiple targets as used hereinmay generally include different types of targets, and/or may alsoinclude a multitude of a singular type of targets at differentconditions, such as, for example, temperature, pH, chemical environment,and/or any other appropriate conditions.

In general, a method for generating functional biomolecules includesobtaining a library, such as a diverse or randomized library, forexample, of biomolecules. Biomolecules may generally include nucleicacids, particularly single-stranded nucleic acids, peptides, otherbiopolymers and/or combinations or modifications thereof. A library ofbiomolecules may include nucleic acid sequences, such as ribonucleicacid (RNA), deoxyribonucleic acid (DNA), artificially modified nucleicacids, and/or combinations thereof. The method for generating functionalbiomolecules further includes contacting the library of biomoleculeswith more than one target, such as, for example, a molecular target,material and/or substance. In general, the members of the library thatdo not bind with some affinity to the more than one target may be washedor otherwise partitioned from the remainder of the library, which mayhave a given level of binding affinity to the more than one target. Theprocess may be repeated to partition the strongest binding members ofthe library. Amplification of the biomolecules may also be utilized toincrease the numbers of the binding members of the library forsubsequent repetitions and for isolation and/or purification of anyfinal products of the process. Embodiments of the SELEX method maygenerally be utilized to achieve the generation of functionalbiomolecules of a given binding affinity, such biomolecules generallyreferred to as aptamers or ligands.

In one exemplary aspect of the invention, generation of functionalbiomolecules may be performed against more than one or multiple targetssimultaneously within a single system, such as the generation offunctional nucleic acid ligands within a single reaction volume. Ingeneral, more than one or a plurality of targets may be disposed withinin a single reaction volume, and a library of biomolecules, such as anucleic acid library, may be applied to the reaction volume. The membersof the library that do not bind to any of the plurality of targets undergiven conditions may then be partitioned, such as by washing. One ormore rounds of binding and partitioning of the members of the librarymay be performed, such as, for example, to obtain a remainder of membersof the library with a given affinity for their targets. The remainingmembers that bind to the plurality of targets of the library may then bemarked and/or tagged, such as to identify the particular target ortargets to which the member(s) of the library binds. The binding membersof the library may then be isolated and, by virtue of the marking ortagging, be matched to a particular target or targets. This is desirableas high capacity, multiplexed identification procedures may save time,expense, and physical space for the process over single targetidentification processes. The present method may also be desirable as itmay be utilized to identify and/or eliminate biomolecules that bind orhave a tendency to bind to multiple targets.

In an exemplary embodiment, a plurality of target molecules are affixedto a substrate within a single reaction volume, such as, for example, byattaching the targets to a substrate of an array. It may generally beappreciated that a single reaction volume may refer to or includemultiple reaction sub-volumes, such as, for example, discrete orsemi-discrete fluid droplets. In general, the targets may be disposedwith multiple copies of each target in clusters or “spots” such that agiven array may have an ordered deposition of targets on the substrate,with each target identifiable by the location of a particular spot onthe substrate. A library of nucleic acids may then be contacted orapplied to the array and the non-binding members of the library may bepartitioned or washed off the array. The binding and washing steps maybe repeated and may also utilize an amplification step to generateadditional copies of any remaining binding members of the library. Thearray may then be marked or tagged with a plurality of identifiers, suchas, for example, a plurality of oligonucleotides which may universallybind through Watson-Crick interactions to the members of the library ofnucleic acids. The marking or tagging may be, for example, accomplishedby manually applying identifiers, such as by pipetting or the like,utilizing microcontact pins, applying membranes/films with identifiers,printing, for example, inkjet printing, and/or other similar taggingmethods, of identifier containing solutions to the array. Theidentifiers may further include a unique or semi-unique sequence whichmay be utilized to correspond to the spots and thus the targets of thearray. For example, a unique or semi-unique identifier sequence may beutilized that identifies each spatial location on an array, such as eachparticular target spot. The identifier may then be associated withand/or attached to the nucleic acid members bound to a particular spot.Thus, the nucleic acids, for example, bound to a particular target spotmay be identified by the sequence of the associated identifier. In someembodiments, the identifiers may further be primers and may be utilizedwith a nucleic acid amplification reaction on the array to generateadditional copies of the bound nucleic acids. The unique or semi-uniqueidentifier sequence may also be incorporated into the members of thelibrary amplified. This may be desirable for associating a given memberwith a target or targets while preserving the particular sequence of themember as the locational identifying sequence is appended to thesequence of the library member. This may be particularly desirable forresolving multiple binders to a single target or members of the librarythat bind to multiple targets.

In general, the starting library of biomolecules, such as nucleic acids,may be the product of at least one round of a previous SELEX protocol.For example, at least one round of SELEX may be performed with a libraryof biomolecules against multiple targets, such as, for example, in asolution. The targets in the solution may be substantially identical tothe targets disposed on an array. This may be desirable as multiplerounds of selection may be performed with a library prior to applyingthe remaining members to an array for marking/tagging. Complex targetarrays may generally be more expensive and/or difficult to make orutilize than solutions of target molecules, so performing only the finalbinding and marking/tagging procedure on the array may be desirable.

In other embodiments, identifiers may be predisposed on the arraysubstrate in substantial proximity to the spots such that they may bindto, for example, nucleic acids bound to the target spots. Theidentifiers may, for example, be covalently attached to the substrate.In some embodiments, the attachments may be controllably breakable orcleavable such that the identifiers may be released from the substratesuch that they may, for example, more easily bind to the bound nucleicacids on the spots.

In further embodiments, identifiers may be synthesized in situ on thearray, such as by light directed in situ nucleic acid synthesis.Appropriately sequenced identifiers may then be synthesized in proximityto particular spots such that the newly synthesized identifiers may bindto the nucleic acids bound to the target spot.

In still other embodiments, identifiers may be disposed and/orsynthesized on a separate substrate, such as a membrane, in a spatialdisposition that substantially matches the spatial disposition of spotson the array, i.e. the identifiers may be arranged such that they may bereadily superimposed onto the target spots on the array. The identifiersubstrate may then be contacted with the array with locational matchingof the spots with identifiers. The identifiers may then bind to thenucleic acids bound to the target spots. Any appropriate method offacilitating binding may be utilized, such as, for example, actions todrive migration of the identifiers to the array, such as capillaryaction, electrophoresis, pressure, gravitational settling, and/or anyother appropriate method or combination thereof. The separate substratemay also be soluble, erodible, substantially permeable to theidentifiers, and/or otherwise adapted for facilitating migration of theidentifiers to the array.

In yet still other embodiments, the array substrate may be physicallydivided and/or partitioned for separate collection of the, for example,nucleic acids bound to the spots. The spots may, for example, also becontrollably removable from the substrate such that they may beindividually recovered and sorted.

In still yet other embodiments, identifiers may be disposed and/orsynthesized on a separate substrate, such as a membrane, in a spatialdisposition that substantially matches the spatial disposition of spotson the array, i.e. the identifiers may be arranged such that may bereadily superimposed onto the target spots on the array. The separatesubstrate may be kept separately while the array substrate maybephysically divided and/or partitioned for separate collection of thenucleic acids bound to the spots. In this manner, the location of thedifferent nucleic acids maybe maintained even when the array substrateis no longer intact, if the locations are of value. The identifiers mayalso be selectively applied to particular locations on the array and/orapplied in a particular order or in groups.

In some embodiments, identifiers may only be applied to spots with boundnucleic acids. The spots with bound nucleic acids may be detected, forexample, by detecting the presence of nucleic acids, such as by applyingnucleic acid binding dyes, such as SYBR dyes, ethidium bromide and/orother appropriate dyes. The members of the nucleic acid library may alsoinclude detectable portions, such as, for example, fluorescent moieties,radioactive tags and/or other appropriate detectable portions.

In some embodiments, the identifiers may be applied to the bound nucleicacids together with other materials, such as for example, components ofa nucleic acid amplification reaction, a nucleic acid ligation reaction,photo-linking reagents, and/or any other appropriate material, such asthose materials that may facilitate attachment or association of theidentifiers to the bound nucleic acids.

In yet another embodiment, identifiers may be ligated to the, forexample, bound nucleic acids. For example, a nucleic acid ligase may beutilized to covalently link an identifier sequence to the bound nucleicacid. Further nucleic acid fragments may be utilized to facilitateligase action, such as appropriate complementary fragments that may aidthe formation of a substantially double-stranded nucleic acid complexcompatible with a ligase. For another example, photo-ligation may beused to attach the identifiers to the, for example, bound nucleic acids.Photo-ligation may be especially useful when certain substrates areused. For example, macro-porous substrates.

In general, methods may be applied that may facilitate binding or otherinteractions between the identifiers and the, for example, nucleic acidsbound to the spots. For example, the temperature may be increased todissociate the nucleic acids from the spots. The temperature maysubsequently be lowered such that, for example, base pairing may occurbetween the nucleic acids and the identifiers. Further in general, itmay be desirable to apply the identifiers in a manner that physicallyseparates and/or isolates the individual target spots such thatcross-marking due to identifier diffusion/migration may be minimized.For example, the identifiers may be applied in individual fluid dropletssuch that there is no continuous fluid contact between individualidentifier containing fluids. For further example, the substrate of thearray may be absorbent and/or porous such that the identifiers may beabsorbed into the substrate material. The substrate material may alsoblock lateral diffusion while allowing vertical diffusion, such thatidentifiers may be applied and absorbed into the substrate whileminimizing diffusion across the plane of the substrate, such as to othertarget spots.

In a further embodiment, a method for generating functional biomoleculesincludes obtaining a library of peptide sequences and contacting thelibrary with a plurality of targets. In some exemplary embodiments, thepeptide sequence may be tagged, linked, marked and/or otherwiseassociated with a nucleic acid sequence. The nucleic acid sequence maybe, for example, representative of the sequence of the peptide. Forexample, the nucleic acid may substantially encode the peptide sequence.Also for example, the nucleic acid may be a unique or semi-uniqueidentifier sequence. The nucleic acid sequence may then be utilized tobind another identifier, as described above, such that a peptide boundto a target may be tagged or marked as to which target it bound.

In an exemplary embodiment, a bacteriophage (phage) may be generatedthat includes a peptide sequence of interest in its protein coat. Thephage may further include a nucleic acid sequence that may berepresentative of the peptide sequence within the nucleic acid of thephage. The phage may then be contacted with a plurality of targets, asabove. This may generally be referred to as phage display. Non-bindingphages may be washed and/or partitioned, while binding phages may betagged or marked with identifiers, as above. As phage nucleic acids aregenerally contained within the protein coat of the phage, the nucleicacid may generally be exposed for binding to the identifier. Forexample, the phage may be heated such that the protein coat denaturesand/or disassembles such that the nucleic acid is exposed. Theidentifier may also be introduced into the phage, such as byelectroporation, electrophoresis, and/or any other appropriate method.

Other methods of peptide selection may include, but are not limited to,mRNA display, ribosome display, and/or any other appropriate peptidedisplay method or combination thereof.

In another aspect of the invention, methods for handling and sorting theresultant sequences of a multiplexed binding process are provided. Insome embodiments, the sequences may be sorted by identifier sequences toestablish which target or targets the sequence bound. The sequences mayfurther be compared, aligned and/or otherwise processed to identifyfeatures, characteristics and/or other useful properties, relationshipsto each other, and/or target properties.

In a further aspect of the invention, methods for monitoring and/orcontrolling the diversity of the library of biomolecules may beutilized. For example, too few rounds of selection may result in abiomolecule pool with too many weak binding members while too manyrounds of selection may result in only a few binding members, such asmembers corresponding to only a few targets rather than memberscorresponding to all of the targets present. In one embodiment, Cotanalysis may be employed to measure and/or monitor the diversity of thelibrary of biomolecules through multiple rounds of selection. Cot, orConcentration x time, analysis measures the annealing time of particularoligonucleotides while in solution with other nucleic acids, such as themembers of the library of biomolecules. In general, the annealing timewill be faster the lower the diversity of the library.

The present invention together with the above and other advantages maybest be understood from the following detailed description of theembodiments of the invention illustrated in the drawings.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates an embodiment of a multiple target array;

FIG. 2 illustrates the application of a library of biomolecules to atarget array;

FIG. 2a illustrates the binding of members of a library of biomoleculesto a target spot;

FIGS. 3 and 3 a illustrate embodiments of biomolecule library members;

FIG. 3b illustrates an embodiment of an identifier;

FIG. 4 illustrates the tagging of a library member bound to target withan identifier;

FIG. 4a illustrates a tagged library member product;

FIG. 5 illustrates a target spot with nearby identifiers on a substrate;

FIG. 5a illustrates the application of an identifier sheet to a targetarray;

FIGS. 6, 6 a, 6 b and 6 c illustrate embodiments of identifiers andligation of identifiers to a library member;

FIGS. 7 and 7 a illustrate phage display for a target;

FIG. 7b illustrates an mRNA display fusion product;

FIG. 7c illustrates a ribosome display fusion product;

FIG. 8 illustrates an example of a histology section target; and

FIG. 9 illustrates an example of binding of an aptamer beacon to atarget molecule.

DETAILED DESCRIPTION OF THE INVENTION

The detailed description set forth below is intended as a description ofthe presently exemplified methods, devices, and compositions provided inaccordance with aspects of the present invention and is not intended torepresent the only forms in which the present invention may be practicedor utilized. It is to be understood, however, that the same orequivalent functions and components may be accomplished by differentembodiments that are also intended to be encompassed within the spiritand scope of the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which this invention belongs. Although any methods, devicesand materials similar or equivalent to those described herein can beused in the practice or testing of the invention, the exemplifiedmethods, devices and materials are now described.

The present invention relates functional ligands to target molecules,particularly to functional nucleic acids and modifications thereof, andto methods for simultaneously generating, for example, numerousdifferent functional biomolecules, particularly to methods forgenerating numerous different functional nucleic acids against multipletarget molecules simultaneously. The present invention further relatesto functional ligands which bind with affinity to target molecules, moreparticularly to functional ligands with binding affinity to targetmolecules such as drug compounds, such as levamisole and cocaine, whichmay also include their derivatives, isomers and/or breakdown products.The present invention further relates to methods for simultaneouslygenerating different functional biomolecules, particularly to functionalnucleic acids, that bind with functional activity to anotherbiomolecule, such as a receptor molecule. Functional ligands,particularly functional nucleic acids, of the present invention aregenerally artificial, non-naturally occurring sequences designed and/orselected for specific and/or high affinity binding to target moleculessuch as drug compounds, such as levamisole and cocaine. Non-naturallyoccurring sequences of functional nucleic acids, such as aptamers, mayalso be useful by interacting with a target molecule in a manner notpresent in naturally occurring systems or situations, such as by, forexample, not being already present or having a pre-existing function ina naturally occurring setting.

In general, a method for generating functional biomolecules includesobtaining a library, such as a diverse or randomized library, ofbiomolecules. Biomolecules may generally include nucleic acids,particularly single-stranded nucleic acids, peptides, other biopolymersand/or combinations or modifications thereof. A library of biomoleculesmay include nucleic acid sequences, such as ribonucleic acid (RNA),deoxyribonucleic acid (DNA), artificially modified nucleic acids, and/orcombinations thereof. In general, modified nucleic acid bases may beutilized and may include, but are not limited to,2′-Deoxy-P-nucleoside-5′-Triphosphate, 2′-Deoxyinosine-5′-Triphosphate,2′-Deoxypseudouridine-5′-Triphosphate, 2′-Deoxyuridine-5′-Triphosphate,2′-Deoxyzebularine-5′-Triphosphate,2-Amino-2′-deoxyadenosine-5′-Triphosphate,2-Amino-6-chloropurine-2′-deoxyriboside-5′-Triphosphate,2-Aminopurine-2′-deoxyribose-5′-Triphosphate,2-Thio-2′-deoxycytidine-5′-Triphosphate,2-Thiothymidine-5′-Triphosphate, 2′-Deoxy-L-adenosine-5′-Triphosphate,2′-Deoxy-L-cytidine-5′-Triphosphate,2′-Deoxy-L-guanosine-5′-Triphosphate,2′-Deoxy-L-thymidine-5′-Triphosphate, 4-Thiothymidine-5′-Triphosphate,5-Aminoallyl-2′-deoxycytidine-5′-Triphosphate,5-Aminoallyl-2′-deoxyuridine-5′-Triphosphate,5-Bromo-2′-deoxycytidine-5′-Triphosphate,5-Bromo-2′-deoxyuridine-5′-Triphosphate,5-Fluoro-2′-deoxyuridine-5′-Triphosphate, 5-Trifluoromethyl-2-deoxyuridine-5′-Triphosphate, and/or any other appropriate modifiednucleic acid base. It may generally be understood that the nucleosidetriphosphates (NTPs) listed above may generally refer to any appropriatephosphate of the modified base, such as additionally, for example,monophosphates (NMPs) or diphosphates (NDPs) of the base. The method forgenerating functional biomolecules further includes contacting thelibrary of biomolecules with at least one target, such as, for example,a molecular target, material and/or substance. In general, the membersof the library that do not bind with some affinity to the target may bewashed or otherwise partitioned from the remainder of the library, whichmay have a given level of binding affinity to the target. The processmay be repeated to partition the strongest binding members of thelibrary. Amplification of the biomolecules may also be utilized toincrease the numbers of the binding members of the library forsubsequent repetitions and for isolation and/or purification of anyfinal products of the process. Embodiments of the SELEX method maygenerally be utilized to achieve the generation of functionalbiomolecules of a given binding affinity. The basic SELEX protocol andaptamers are described in U.S. Pat. No. 5,270,163, entitled “Methods foridentifying nucleic acid ligands,” the entire contents of which arehereby incorporated by reference.

In one exemplary aspect of the invention, generation of functionalbiomolecules may be performed against multiple targets simultaneouslywithin a single system, such as the generation of functional nucleicacid ligands within a single reaction volume. In general, a plurality oftargets may be disposed within in a single reaction volume and a libraryof biomolecules, such as a nucleic acid library, may be applied to thereaction volume. The targets may be, for example, proteins, cells, smallmolecules, biomolecules, and/or combinations or portions thereof. Themembers of the library that do not bind to any of the plurality oftargets under given conditions may then be partitioned, such as bywashing. The remaining members of the library may then be marked and/ortagged, such as to identify the particular target or targets to whichthe member of the library binds. The binding members of the library maythen be isolated and, by virtue of the marking or tagging, be matched toa particular target or targets. This may be desirable as high capacity,multiplexed identification procedures may save time, expense, andphysical space for the process over single target identificationprocesses. The present method may also be desirable as it may beutilized to identify and/or eliminate molecules that bind to multipletargets.

Functional ligands to levamisole, cocaine and related molecules, withoutlimitation and without being bound to any particular theory, may beutilized for detection, quantification, and/or other diagnosticapplications, such as for detecting levamisole in body fluids ortissues, such as blood or saliva, such as for identifying levamisole asan adulterant in drugs, such as cocaine, or for quantification inclinical poisoning situations by levamisole. For further example,detection of cocaine may be utilized in bodily fluids or tissue forascertaining the use of cocaine by a person. For example, and withoutbeing bound to any particular theory, a functional ligand such as anucleic acid aptamer to levamisole may be modified with a moiety thatparticipates in fluorescence energy transfer (FRET) or other pairedsignal interaction with another moiety attached a nucleic acid thathybridizes to the aptamer. The aptamer may generally be utilized to bindlevamisole to cause the aptamer to dehybridized from the other nucleicacid and/or otherwise increase the distance between the moieties suchthat the FRET or other signal interaction is altered in a detectableway, indicating the presence of the target protein(s). Other forms ofdetection may also utilize the functional ligands, such aselectrochemical sensors, gold nanoparticle assays, enzyme linked aptamersorbent assays (ELASA), pull down assays (immunoprecipitation),microplate/well assays, lateral flow assays and/or any other appropriateform of detection.

In an exemplary embodiment, a plurality of target molecules are affixedto a substrate within a single reaction volume, such as, for example, byattaching the targets to a substrate of an array. As illustrated in FIG.1, the targets may be disposed with multiple copies of each target, suchas target molecules, in clusters or “spots” 110 on the substrate 102 ofan array 100 such that a given array 100 may have an ordered depositionof targets on the substrate 102, with each target identifiable by thelocation of a particular spot 110 on the substrate 102. Each spot 110may be a unique target or there may be multiple spots 110 of at leastone target on a given array 100. In general, high content target arrays,such as high content protein or antibody arrays or other forms of arraysrecognized as high content or high density for interrogating a highnumber of different targets at once may be utilized. For example, humanprotein microarrays, such as ProtoArray® from ThermoFisher containingover 9,000 unique proteins in spotted format, may be utilized. A library200 of, for example, nucleic acids 202 may then be applied A to array100, as illustrated in FIG. 2. Particular members 204 of the library 200may then bind to target spots 110, such as illustrated in FIG. 2 a. Thenon-binding members 206 of the library 200 may be partitioned or washedoff the array 100. The binding and washing steps may be repeated and mayalso utilize an amplification step to generate additional copies of anyremaining binding members 204 of the library 200. The array 100 may thenbe marked or tagged with a plurality of identifiers, such as, forexample, a plurality of oligonucleotides which may universally bindthrough Watson-Crick interactions to the members of the library of, forexample, nucleic acids. In one embodiment, each member 202 of thelibrary 200 may include a potential binding sequence 202 a and at leastone conserved region 202 b which may bind an identifier oligonucleotide,such as illustrated in FIG. 3. A further conserved region 202 c may alsobe included to facilitate priming for amplification or extensionreactions, such as Polymerase Chain Reaction (PCR), as illustrated inFIG. 3 a. In general the conserved regions 202 b, 202 c may flank thepotential binding sequence 202 a, such as to facilitate priming foramplification. An identifier 302 may then include a unique orsemi-unique sequence 302 a, such as illustrated in FIG. 3 b, which maybe utilized to correspond to the spots 110 and thus the targets of thearray 100 by location of the a spot 110 on the substrate 102. Theidentifiers 302 may further include conserved region 302 b which maybind to the conserved region 202 b of the library members 202 byWatson-Crick base pairing. The identifiers 302 may also include afurther conserved region 302 c which may facilitate priming foramplification. The identifiers 302 may be, for example, applied to thespots 110 by printing, for example, inkjet printing, using micro-contactpins, and/or otherwise applying solutions containing identifiers 302 tothe substrate 102 of the array 100, such as, for example, by pipettingor the like, onto the spots 110. A library member 202 bound to a targetspot 110 may then be tagged with an identifier 302 via base pairing B atregions 202 b, 302 b, as illustrated in FIG. 4. Thus, the nucleic acids202 bound to a particular target spot 110 may be identified by thesequence 302 a of the identifier 302. In an exemplary embodiment,nucleic acid amplification or extension, such as PCR, may be utilized togenerate copies of the members 202 bound to the spots 110, incorporatingthe identifier sequence 302 a (or more its complementary sequence) intothe product 203, as illustrated in FIG. 4 a. This may be desirable forassociating a given member 202 with a target or targets 110 whilepreserving the particular sequence of the member 202. This may beparticularly desirable for resolving multiple binders to a single targetor members of the library that bind to multiple targets. Subsequentamplifications may utilize primers for the sequences 202 c, 302 c suchthat only the products 203 containing both the sequences 202 a, 302 aare amplified. It may be understood that references to nucleic acidsequences, such as above, may generally refer to either a particularsequence or the corresponding complementary nucleic acid sequence. Ingeneral, it may be desirable for single droplets and/or otherwiseseparated volumes of solutions containing identifiers 302 for each spot110 on the array 100 such that the possibility of mistagging may bereduced.

In one aspect, the identifiers may be printed on all the targets. Inanother aspect, the identifiers may be printed only on targets withbound biomolecules.

In another embodiment, a histology section, such as the section 110″ onsubstrate 102″ of histology slide 100″ in FIG. 8, may be utilized as atarget set. The section 110″ may be, for example, a tissue section, acell mass, and/or any other appropriate biological sample which maygenerally have structurally significant features. As with the array 100,a library of biomolecules, such as nucleic acids, may be applied whichmay bind to specific locations on the section 110″, the locations onwhich may, for example, represent separate targets to generate affinitybinding nucleic acids. Identifiers may then be disposed on the slide100″ as described above, or as in the embodiments below, such thatidentifiers may be utilized to correspond to specific features of thesection 110″.

In other embodiments, identifiers may be predisposed on the arraysubstrate in substantial proximity to the spots, such as illustratedwith identifiers 302 disposed on substrate 102 in proximity to spot 110in FIG. 5, such that they may bind to nucleic acids bound to the targetspots. The identifiers may, for example, be covalently attached to thesubstrate. In some embodiments, the attachments may be controllablybreakable or cleavable such that the identifiers may be released fromthe substrate such that they may, for example, more easily bind to thebound nucleic acids on the spots.

In further embodiments, identifiers may be synthesized in situ on thearray, such as by light directed in situ nucleic acid synthesis.Appropriately sequenced identifiers may then be synthesized in proximityto particular spots such that the newly synthesized identifiers may bindto the nucleic acids bound to the spot.

In still other embodiments, identifiers may be disposed and/orsynthesized on a separate substrate, such as a membrane, in a spatialdisposition that matches the spatial disposition of spots on the array.FIG. 5a illustrates an example of an identifier sheet 100′ with membrane102′ which may include identifier spots 110′ which may substantiallycorrespond to target spots 110 of the array 100. The identifier sheet100′ may then be contacted C with the array 100 with locational matchingof the target spots 110 with identifier spots 110′. The identifiers maythen bind to the nucleic acids bound to the target spots. Anyappropriate method of facilitating binding may be utilized, such as, forexample, actions to drive migration of the identifiers to the array,such as capillary action, electrophoresis, pressure, gravitationalsettling, and/or any other appropriate method or combination thereof.

In some embodiments, the membrane may be soluble and/or substantiallyerodible. For example, the membrane may include a film forming and/orsoluble material. Identifiers and/or other materials, such as componentsof a nucleic acid amplification or ligation reaction, may be includedsuch that a film is formed containing the desired materials. Themembrane may then be applied to the substrate and a suitable solvent,such as water or ethanol, may be utilized to dissolve and/or erode thefilm, which may then release the included materials, such as theidentifiers, to the substrate. Suitable materials for the film mayinclude hydrophilic materials including polysaccharides such ascarrageenan, chondroitin sulfate, glucosamine, pullulan, solublecellulose derivatives such as hydroxypropyl cellulose and hydroxymethylcellulose, polyacrylic acid, polyvinyl alcohol, polyethylene glycol(PEG), polyethylene oxide (PEO), ethylene oxide-propylene oxideco-polymer, polyvinylpyrrolidone (PVP), polycaprolactone,polyorthoesters, polyphosphazene, polyvinyl acetate, andpolyisobutylene.

The membrane may further be adapted to have a desirable rate of erosionand/or dissolution. The rate may be modified by the inclusion ofhydrophobic and/or less soluble additives. Suitable materials mayinclude, but are not limited to, those from the family of quaternaryammonium acrylate/methacrylate co-polymers, (Eudragit RS), cellulose andits lower solubility derivatives, such as butyl cellulose, hydroxybutylcellulose and ethylhydroxyethyl cellulose, high molecular weight PEG orPEO or a combination thereof.

In yet still other embodiments, the array substrate may be physicallydivided and/or partitioned for separate collection of the nucleic acidsbound to the spots. The spots may, for example, also be controllablyremovable from the substrate such that they may be individuallyrecovered and sorted. The array itself may also be perforated and/orotherwise easily and/or conveniently partitionable.

In another embodiment, identifiers may be ligated to the bound nucleicacids. For example, a nucleic acid ligase may be utilized to covalentlylink an identifier sequence to the bound nucleic acid. In general,nucleic acid ligases are enzymes that covalently join two nucleic acidsby catalyzing the formation of phosphodiester bonds at the ends of thephosphate backbone of the nucleic acids. Examples of appropriate nucleicacid ligases may include, but are not limited to, E. coli DNA ligase, T4DNA ligase, T4 RNA ligase, strand break DNA repair enzymes, and/or anyother appropriate ligase, modified enzyme, and/or a combination thereof.In general the ligase utilized may be selected based on the form ofligation performed, such as ligation of blunt ends, compatible overhang(“sticky”) ends, single stranded DNA, singe stranded RNA and/or anyother form of ligation. Further in general, the steps in ligating twonucleic acids together is a one step process that may be carried out ator near room temperature. Further nucleic acid fragments may be utilizedto facilitate ligase action, such as appropriate complementary fragmentsthat may aid the formation of a substantially double-stranded nucleicacid complex compatible with a ligase. In general, double strandedligation may be employed and may utilize substantially compatibleoverhang fragments to facilitate ligation, or also blunt end ligationmay be utilized, such as with either the nucleic acid end or theidentifier having a phosphorylated end while the other isunphosphorylated for ligation. Single stranded ligation may also beemployed.

Photo ligation may also be employed. Photo ligation may, for example,include covalently linking adjacent nucleic acids by application ofelectromagnetic energy, such as ultraviolet or visible light. Couplingagents may also be utilized to facilitate the formation of covalentlinkages.

In some embodiments, dyes may be included into the identifiers. In oneaspect, the identifiers may be doped with dyes. In another aspect, theidentifier solutions may be mixed with dyes. According to oneembodiment, the dyes may be photosensitive and may be fluorescent.According to another embodiment, the dyes may be photosensitive and maybe phosphorescent.

The substrates used may be glass, ceramic or polymeric, as long as theirsurfaces promote adhesion between the substrates and the targets.Polymers may include synthetic polymers as well as purified biologicalpolymers. The substrate may also be any film, which may be non-porous ormacroporous.

The substrate may be generally planar and may be of any appropriategeometry such as, for example, rectangular, square, circular,elliptical, triangular, other polygonal shape, irregular and/or anyother appropriate geometry. The substrate may also be of other forms,such as cylindrical, spherical, irregular and/or any other appropriateform.

Appropriate ceramics may include, for example, hydroxyapatite, alumina,graphite and pyrolytic carbon.

Appropriate synthetic materials may include polymers such as polyamides(e.g., nylon), polyesters, polystyrenes, polyacrylates, vinyl polymers(e.g., polyethylene, polytetrafluoroethylene, polypropylene andpolyvinyl chloride), polycarbonates, polyurethanes, poly dimethylsiloxanes, cellulose acetates, polymethyl methacrylates, ethylene vinylacetates, polysulfones, nitrocelluloses and similar copolymers. Thesesynthetic polymers may be woven or knitted into a mesh to form a matrixor similar structure. Alternatively, the synthetic polymer materials canbe molded or cast into appropriate forms.

Biological polymers may be naturally occurring or produced in vitro byfermentation and the like or by recombinant genetic engineering.Recombinant DNA technology can be used to engineer virtually anypolypeptide sequence and then amplify and express the protein in eitherbacterial or mammalian cells. Purified biological polymers can beappropriately formed into a substrate by techniques such as weaving,knitting, casting, molding, extrusion, cellular alignment and magneticalignment. Suitable biological polymers include, without limitation,collagen, elastin, silk, keratin, gelatin, polyamino acids,polysaccharides (e.g., cellulose and starch) and copolymers thereof.

Any suitable substrate may be susceptible to adhesion, attachment oradsorption by targets. The susceptibility may be inherent or modified.In one example, the surfaces of substrates may be susceptible toadhesion, attachment or adsorption to proteins. In another example, thesurfaces of substrates may be susceptible to adhesion, attachment oradsorption to proteins and not to nucleic acids.

In one exemplary embodiment, a glass substrate may have a layer orcoating of a material that promotes adhesion with targets, such asproteins, materials that maybe charged, such as those that arepositively charged, for binding target materials. Examples of chargedmaterials include cellulosic materials, for example, nitrocellulose,methylcelluose, ethylcellulose, carboxymethyl cellulose, hydroxyethylcellulose, methylhydroxypropyl cellulose; epoxies, PVDF (polyvinylidenefluoride); partially or fully hydrolyzed poly(vinyl alcohol);poly(vinylpyrrolidone); poly(ethyloxazoline); poly(ethyleneoxide)-co-poly(propylene oxide) block copolymers; polyamines;polyacrylamide; hydroxypropylmethacrylate; polysucrose; hyaluronic acid;alginate; chitosan; dextran; gelatin and mixtures and copolymersthereof.

In another exemplary embodiment, if the substrate is not susceptible forattachment by charged materials, or may be susceptible only forattachment by wrongly charged materials, some areas of the substrate mayhave adhesives, binding agents, or similar attached, adsorbed or coatedthereon. Examples of adhesives may include any suitable adhesives thatbind the charged materials.

The targets may be present on the substrate discretely or in clusters.The distance between the discrete targets may be close or may be farapart and may usually be of different targets. Clusters may be used formultiple spots of a single target.

In one embodiment, the substrate may be macroporous. Macroporoussubstrates may be desirable, for example, if the different targets arevery close together. When the targets are close by, there may not besufficient distance between different targets to distinguish whichtarget a biomolecule may be binding to. Closely packed targets mayincrease the efficiency of the generating of biomolecules. A macroporoussubstrate may be suited for balancing between efficiency and separation.For a macroporous substrate, the walls of the pores may be sufficient toseparate even closely packed targets if the pores are large enough toenable the binding process to occur within the pores.

Also, for macroporous substrates, the pores may have an average diametergreater than the average size of the target material such that thetarget material may enter or partly enter the pores to anchor. Hydrogelsmay also be useful for binding or anchoring targets to the pores.Hydrogels may also fill the pores under fluid conditions and present asmooth surface for fluid flow while at the same time may keep the fluidfrom flowing through the pores.

The plurality of targets may be arranged in any appropriate manner suchas, for example, in circular or elliptical spots, square or rectangularspots, stripes, concentric rings and/or any other appropriatearrangement on the subject.

According to one exemplary embodiment, the substrate may be at ambienttemperature throughout.

According to another exemplary embodiment, the substrate may include atemperature affecting system that generally produces at least onedesired temperature on the surface of the substrate and the adjacentfluid. The desired temperature may facilitate the biomolecule generatingprocess.

According to a further exemplary embodiment, the substrate may include atemperature affecting system for producing a range of desiredtemperatures on the surface of the substrate and the adjacent fluid.This may be particularly useful when employing a set of targets having asignificant range of, for example Tms, or melting temperatures. In oneembodiment, the system may include a plurality of temperature affectingdevices that are in thermal communication with the substrate. Theplurality of devices may generally be disposed such that they may eachproduce a desired temperature in a given locality on the surface of thesubstrate. The set of targets may also be distributed on the surface ofthe substrate such that the temperature at the location of a target issubstantially at the Tm of the target.

Temperature affecting devices may be any appropriate device that maysubstantially produce a desired temperature on a substrate and mayinclude, but are not limited to, thermoelectric devices such as Peltierjunction devices, semiconductor heating devices, resistive heatingdevices, inductive heating devices, heating/cooling pumps,electromagnetic radiation sources and/or any other appropriate devices.Temperature may also be affected by other systems, such as, for example,fluid flows including, but not limited to, water flows, air flows,and/or any other appropriate fluid flows.

In an exemplary embodiment, a plurality of Peltier junction devices maybe utilized to generate desired temperatures at localities on thesurface of the substrate. Peltier junction devices are particularlyuseful since they are able to both heat and cool using electricalcurrent. This enables Peltier junction devices to generate temperaturesabove and below the ambient temperature of a system. They may also beuseful in maintaining given temperature conditions at a steady state byadding and removing heat as necessary from the system.

In general, the placement of the temperature affecting devices maydetermine the temperature profile on the surface of the substrate andthe adjacent fluid in the chamber. The temperature affecting devices maythus be disposed at appropriate positions such that given temperaturesmay be produced and maintained at known positions on the substrate.

The substrate may in general have a given thermal conductivity such thatthe application of at least one temperature affecting device maysubstantially generate a temperature gradient profile on the surface ofthe substrate. In general, the temperature on the surface of thesubstrate may change as a function of the distance from the position ofthe at least one temperature affecting device. Substrate materials witha relatively low thermal conductivity may generally produce highlylocalized temperature variations around a temperature affecting device.Substrate materials with a relatively high thermal conductivity maygenerally produce more gradual variations in temperature over a givendistance from a temperature affecting device. It may be understood thatat steady state, the effect of the thermal conductivity of the substratemay not contribute to the temperature profile of the system.

In some embodiments, at least one temperature affecting device may beutilized to produce a particular temperature gradient profile on thesurface of the substrate. In general, a temperature gradient may begenerated by utilizing at least one temperature affecting deviceproducing a temperature different from the ambient temperature of thesystem. Multiple temperature affecting devices with at least twoproducing different temperatures may be utilized to generate atemperature gradient without reliance on the ambient temperature of thesystem.

The positions and temperatures of multiple temperature affecting devicesmay be utilized to calculate a resulting temperature gradient profile onthe surface of a substrate using standard heat transfer equations. Analgorithm may then be utilized to calculate the optimal positions and/ortemperatures for a plurality of temperature affecting devices to producea desired temperature gradient profile on the surface of a substrate.The algorithm may be, for example, applied using a computationalassisting system, such as a computer and or other calculatory device.This may be performed to tailor a temperature gradient profile to aparticular substrate with a known disposition of targets of known and/orcalculated Tm. Similarly, a set of targets of known and/or calculated Tmmay be arranged on a substrate based on a temperature gradient profile.This may be desirable as placement of a target at a given location on asubstrate may be accomplished more easily than tailoring a temperatureprofile to pre-existing locations of targets on a substrate. In general,a target may be disposed on the substrate at a temperature addresswithin the temperature profile gradient. The temperature address may,for example, be substantially at the Tm of the target during operationof the molecular hybridization system, and/or any other appropriatetemperature.

In another aspect, the molecular hybridization system includes anadjustable system for generating a temperature profile. The adjustablesystem generally includes a plurality of temperature affecting devices,each affecting the temperature at a particular location of a substrate.

Details of the temperature affecting systems may be found in, forexample, U.S. utility patent application Ser. No. 12/249,525, filed onOct. 10, 2008, entitled “METHODS AND DEVICES FOR MOLECULAR ASSOCIATIONAND IMAGING”, the contents of all of which are hereby incorporated byreference.

FIG. 6 illustrates an example of an identifier sequence 302 and acomplement identifier sequence 402. The complement sequence 402 mayinclude a complement identifier region 402 a which may be substantiallycomplementary to identifier region 302 a such that they may base pairbind. The complement sequence 402 may further include a primer region402 c which may also be complementary to primer region 302 c of theidentifier 302. Further, the complement sequence 402 may include acompatible end 402 b which may be compatible with ligation to the end ofanother nucleic acid. As shown in FIG. 6 a, a nucleic acid librarymember 202 may be bound to a spot 110. An identifier 302 and acomplement sequence 402 may then be applied to the member 202 such thatthe identifier 302 binds to the member 202 at region 202 b, 302 b. Thecomplement sequence 402 may bind to the identifier 302 at regions 302a/402 a, 302 c/402 c. The compatible end 402 b may then be ligated tothe end D of the member 202 by an appropriate ligase and/or otherappropriate method. A product 203′, as illustrated in FIG. 6 b, may thenbe generated including the primer region 202 c, binding sequence 202 a,region 202 b, complement identifier region 402 a, and complement primerregion 402 c. The product 203′ may then be amplified, such as with theproduct 203 discussed above in FIG. 4 a. The product 203′ may also begenerated by single-stranded ligation of the member 202 and thecomplement sequence 402, where in general the either the member 202 orthe complement sequence 402 may have a phosphorylated end while theother may be unphosphorylated for end to end ligation.

In another example, as illustrated in FIG. 6 c, a further complementaryfragment 502 may be included that may base pair bind to a complementaryregion 202 d of the nucleic acid library member 202. This may bedesirable as some nucleic acid ligases may generally join doublestranded nucleic acids. The addition of the complementary fragment 502may generally generate a substantially double stranded nucleic acid,such as illustrated spanning from region 302 c to the end ofcomplementary fragment 502. There may further be a double stranded“break” at points D and E. In general, the sizing of the fragments maybe tailored to generate a suitably long stretch of double strandednucleic acid for ligase action. In general, the complementary region 202d may be the same for all members 202 of the library 200 such that thesame complementary fragment 502 may be utilized, such as, for example,convenience, cost and/or ease of use.

In general, methods may be applied that may facilitate binding or otherinteractions between the identifiers and the nucleic acids bound to thespots. For example, the temperature may be increased to dissociate thenucleic acids from the spots. The temperature may subsequently belowered such that, for example, base pairing may occur between thenucleic acids and the identifiers. Temperature changes may also, forexample, denature the target such that the nucleic acids may no longerbind and/or bind with lower affinity to the targets. This may bedesirable in that it may aid in binding of the nucleic acids to theidentifiers.

In a further aspect of the invention, methods for monitoring and/orcontrolling the diversity of the library of biomolecules may beutilized. For example, too few rounds of selection may result in abiomolecule pool with too many weak binding members while too manyrounds of selection may result in only a few binding members, such asmembers corresponding to only a few targets rather than memberscorresponding to all of the targets present. In one embodiment, Cotanalysis may be employed to measure and/or monitor the diversity of thelibrary of biomolecules through multiple rounds of selection. Cot, orConcentration x time, analysis measures the annealing time of particularoligonucleotides while in solution with other nucleic acids, such as themembers of the library of biomolecules. In general, the annealing timewill be faster the lower the diversity of the library.

In one embodiment, a Cot-standard curve for measuring the sequencediversity of the aptamer library at any point during the multiplex SELEXprocess may be utilized. For example, a group of DNA oligonucleotideswith a 5′ - and 3′-constant region of ˜20 bases identical to the initialSELEX library may be utilized. The oligos may then be converted to dsDNAby standard methods. Briefly, after annealing a primer to the oligos,Exo-minus Klenow Taq polymerase (Epicentre, Madison, Wis.) may be usedin conjunction with dNTPs to fill in the ssDNA to create a dsDNA ormixture thereof. Using a standard quantitative PCR thermal cycler, atemperature profile for melting and controlled annealing of each DNAmixture may be programmed. Standard SYBR Green I specific fordouble-stranded DNA (dsDNA) may be utilized to report the amount ofre-annealed dsDNA. At one extreme, the annealing time for a singlesequence will be measured. At the other extreme, the annealing time forthe initial SELEX pool, such as containing approximately 1 nmol ofsequence diversity, may be measured. Annealing times of intermediatediversity may also be measured to establish a very specificCot-standard-curve for the SELEX library. Using this standard curve, atany point during SELEX, the sequence diversity of the evolving libraryof aptamers may be determined by comparison to the curve.

In a further embodiment, a method for generating functional biomoleculesincludes obtaining a library of peptide sequences and contacting thelibrary with a plurality of targets. In some embodiments, the peptidesequence may be tagged, linked, marked and/or otherwise associated witha nucleic acid sequence. The nucleic acid sequence may be, for example,representative of the sequence of the peptide. For example, the nucleicacid may substantially encode the peptide sequence. Also for example,the nucleic acid may be a unique or semi-unique identifier sequence. Thenucleic acid sequence may then be utilized to bind another identifier,as described above, such that a peptide bound to a target may be taggedor marked as to which target it bound.

In an exemplary embodiment, a bacteriophage (phage) may be generatedthat includes a peptide sequence of interest in its protein coat. Thephage may further include a nucleic acid sequence that may berepresentative of the peptide sequence within the nucleic acid of thephage. The phage may then be contacted with a plurality of targets, asabove. This may generally be referred to as phage display. Phagesemployed may include, but are not limited to, M13 phage, fd filamentousphage, T4 phage, T7 phage, γ phage, and/or any other appropriate phage.Non-binding phages may be washed and/or partitioned, while bindingphages may be tagged or marked with identifiers, as above. As phagenucleic acids are generally contained within the protein coat of thephage, the nucleic acid may generally be exposed for binding to theidentifier. For example, the phage may be heated such that the proteincoat denatures and/or disassembles such that the nucleic acid isexposed. The identifier may also be introduced into the phage, such asby electroporation, electrophoresis, and/or any other appropriatemethod.

In FIG. 7, an example of a phage 600 may include a nucleic acid 610which may generally encode, among other things, and be encapsulated by aprotein coat 602, which may contain a binding region for a target 110.The nucleic acid 610 may further include a region 612 which may identifythe phage and/or encode the binding region for a target. A bound phage600, as illustrated in FIGS. 7 and 7 a, may then be heated, disruptedand/or otherwise treated such that an identifier 302 may contact F theregion 612. For example, the protein coat 602 may be broken and/orotherwise disrupted for entry of the identifier 302. In general, anamplification reaction and/or other method, such as those discussedabove, may be utilized to tag, mark and/or otherwise introduceidentifier information to the sequence of region 612. Further ingeneral, the identifier 302 and region 612 may incorporate any, all or acombination of the elements discussed above in regards to nucleic acidlibrary members, identifiers and/or other nucleic acid fragments. Asalso discussed above, the phage 600 may also be physically removedand/or partitioned in a manner that may preserve the identity of thetarget 110 the phage 600 was associated.

In other embodiments, other methods of incorporating and/or linkingnucleic acids to peptides may be utilized, such as, for example, mRNAdisplay, ribosome display, and/or any other appropriate method. Ingeneral, in mRNA display, as illustrated in FIG. 7 b, a fusion product600′ of a messenger RNA (mRNA) 610′ may be linked to a peptide 602′ thatthe mRNA 610′ encodes, such as with a puromycin-ended mRNA 612′ whichmay generally cause fusion of the mRNA 610′ to the nascent peptide 602′in a ribosome, which may then be contacted with targets such asdescribed above with phage display. Also in general, in ribosomedisplay, as illustrated in FIG. 7 c, a fusion product 600″ of a modifiedmRNA 610″ may be utilized that codes for a peptide 602″, but lacks astop codon and may also incorporate a spacer sequence 612″ which mayoccupy the channel of the ribosome 620″ during translation and allow thepeptide 602″ assembled at the ribosome 620″ to fold, which may result inthe peptide 602″ attached to the ribosome 620″ and also attached to themRNA 610″. This product 600″ may then be contacted with targets such asdescribed above with phage display. Other methods may include, but arenot limited to, yeast display, bacterial display, and/or any otherappropriate method.

In another aspect of the invention, methods for handling and sorting theresultant sequences of a multiplexed binding process are provided. Insome embodiments, the sequences may be sorted by identifier sequences toestablish which target or targets the sequence bound. The sequences mayfurther be compared, aligned and/or otherwise processed to identifyfeatures, characteristics and/or other useful properties, relationshipsto each other, and/or target properties. For example, it may be expectedthat multiple aptamer sequences bound to a single target may potentiallyshare sequence motifs and/or other common features which may be at leastpartially elucidated by sequence sorting and/or comparison. Specificbinding affinities of resultant sequences may also be determined throughaffinity assays. In some embodiments, surface plasmon resonance may beutilized to determine binding of an aptamer to a target. For example,sensors which monitor the refractive index of a surface bound to atarget may be utilized, where the refractive index may change as aresult of binding of an aptamer to the target. In general, aside fromstandard sequencing methods, parallel sequencing methods, such as, forexample, massively parallel sequencing such as 454 Clonal Sequencing(Roche, Branford, Conn.), massively parallel clonal array sequencing,Solexa Sequencing (Illumina, San Diego, Calif.), and/or any otherappropriate sequencing method may be employed.

Aptamers may also be utilized to create molecular beacons which mayfluoresce and/or otherwise produce a detectable signal when the aptamerbinds to its target. Aptamers typically undergo a conformational changewhen binding a target and this conformational change may be utilized tomodulate the activity of other molecules or components of a molecule,such as modulating the distance between a fluorophore (fluor) and aquencher. In general, an aptamer beacon may include an aptamer with afluor and a quencher attached to the 5′ and 3′ ends, respectively, orvice versa. The aptamer in its unbound state may generally be designedto keep the fluor and quencher in proximity such that quenching of thefluor occurs and thus little or no fluorescent signal produced. Linkersand/or stem structures may also be utilized with the base aptamer toproduce this quenching effect in the unbound state. Such linkers and/orstem structures to produce “beacon” structures in nucleic acids aregenerally well known and are standard laboratory techniques. When theaptamer binds to its target, its conformational change upon binding maythen generally cause spacing of the fluor and quencher such that thefluor may undergo fluorescence without quenching by the quencher, andsuch fluorescence may then be detected as a signal to indicate bindingof the target to the aptamer. FIG. 9 illustrates the conformationalchanges of an unbound aptamer beacon structure 700 when binding to atarget molecule 110 at the aptamer portion 702, showing the fluor 704and quencher 706 in an initial proximity resulting in quenching A and afluorescence emitting B conformation after binding the target molecule110 to yield bound conformation 700′.

Aptamers may also be selected and/or designed to exhibit large,detectable and/or specific switching conformational changes when bindingto a target molecule. In general, aptamers frequently exhibitinduced-fit folding behavior, where the aptamer may be largelyunstructured in solution and may undergo significant compaction and/orstructural stabilization upon binding its target molecule. In someembodiments, aptamers may be selected and/or designed such that aportion of the aptamer may hybridize to another nucleic acid, such as aprimer, anchoring oligo and/or other nucleic acid with a complementarysequence, and may dehybridize from such nucleic acid when the aptamerbinds to its target molecule, such as to release the aptamer from suchhybridized nucleic acid.

Example of Multiplexed Selex Protocol

As a demonstration of parallel, de novo selection of aptamers againstmultiple targets, a combinatorial DNA library containing a corerandomized sequence of 40 nts flanked by two 20 nt conserved primerbinding sites is used as the starting point for an aptamer pool. Theprimer sequences are designed and optimized using Vector NTI's(Invitrogen) oligo analysis module. Typically, such a library isexpected to contain approximately 10¹⁵ unique sequences. The primerbinding sites are used to amplify the core sequences during the SELEXprocess. The single stranded DNA pool dissolved in binding buffer isdenatured by heating at 95° C. for 5 min, cooled on ice for 10 min andexposed to multiple protein targets fixed onto a nitrocellulose coatedglass slide (e.g., Whatman).

Example of DNA Library Selex

An example DNA library consists of a random sequence of 40 nucleotidesflanked by conserved primers. In the first round of SELEX, 500 pmol ofthe ssDNA pool is incubated with each slide in binding buffer (PBS with0.1 mg/ml yeast tRNA and 1 mg/ml BSA) for 30 minutes at 37° C. The slideis then washed in 1 ml of binding buffer for one minute. To elutespecifically bound aptamers the slide is heated to 95° C. in bindingbuffer. The eluted ssDNA is subsequently be precipitated using a highsalt solution and ethanol. After precipitation, the aptamer pellet isresuspended in water and amplified by PCR with a 3′-biotin-labeledprimer and a 5′-fluorescein (FITC)-labeled primer (20 cycles of 30 secat 95° C., 30 sec at 52° C., and 30 sec at 72° C., followed by a 10 minextension at 72° C.). The selected FITC-labeled sense ssDNA is separatedfrom the biotinylated antisense ssDNA by streptavidin-coated Sepharosebeads (Promega, Madison, Wis.) for use in the next round. Alternatively,“asymmetric PCR” may be utilized for generating a large excess of anintended strand of a PCR product in SELEX procedures. Alsoalternatively, the undesired strand may be digested by γ-exonuclease,such as, for example, when a phosphorylated PCR primer is employed.

The labeling of individual aptamers with fluorescein isothiocyanate(FITC) facilitates the monitoring of the SELEX procedure. FITC is alsocompatible with scanning in the green (cy3) channel of standardmicroarray scanners. The sense primer used to amplify the ssDNA aptamersafter each round of selection is fluorescently labeled, resulting influorescently labeled aptamers. The protein spottednitrocellulose-coated slides are scanned in a microarray scanner.Alternatively, proteins may be spotted on epoxy-coated glass slides.While epoxy slides may have less protein binding capacity than 3-Dnitrocellulose pads, it has been observed that there may be lessnon-specific binding of nucleic acid aptamer pools to the background ofthe slide (blocked or not). Blocking may be employed to reducebackground fluorescence.

In each round of the SELEX process, the slide is incubated for 30 min at37° C. to allow binding of the aptamers to their targets. The slides arethen washed in binding buffer before the specifically bound DNAs areeluted by heating the slide at 95° C. in 7M urea. Nucleic acids from theeluate are phenol-chloroform purified and precipitated, and theconcentrated single stranded DNA molecules will be amplified by PCR. Inorder to increase stringency throughout the SELEX process, the washesare gradually increased in volume (from approximately 1-10 ml). After agiven point in the selection, such as, for example, after the finalround of selection, the aptamers may be tagged, marked and/orpartitioned.

Example of in Situ Hybridization of Identifiers

An example of in situ hybridization of identifiers to aptamers wasperformed with short, ssDNA sequence tags to the 3′ end of aptamersbound to their protein target. These synthetic ssDNA tagoligonucleotides consists of three regions, as illustrated in FIG. 3bwith identifier 302: (i) the C2 region, region 302 c of the identifier302, at the 3′ end of the oligonucleotide consists of a 17-20 nucleotidesequence complementary to a corresponding region on all of the usedaptamers, (ii) the C1 region, region 302 b at the 5′ end of theoligonucleotide 302 contains a 17-20 nt primer binding site, used duringthe amplification of the tag:aptamer hybrid, prior to sequencing and(iii) a variable region 302 a in the center of the tag oligonucleotide(V) that serves a as a unique identifier for each locus on the glassslide surface. A variable sequence of 8 nucleotides will allow 48(65,536) unique sequences to be generated, sufficient for many complexprotein arrays (8000 samples) on the market.

As outlined above, after the final round of the SELEX procedure(typically, round 10) the specific aptamers are bound to their proteintargets, fixed to a glass slide. While the 40 nt core sequence of eachaptamer are unique, its terminal sequences have not been subject to anykind of selection during the procedure. After each round of binding totheir protein targets, the aptamers were amplified using conservedprimers, requiring the maintenance of corresponding regions at theirdistal ends (P1, P2). The 3′-region of each aptamer, for instance, canthus serve as a binding site (via standard hybridization) for the C2region of the proposed tag oligonucleotide. Given the unique variablesequence (V) of each tag oligonucleotide, each aptamer will now betagged with a sequence that can be traced back to the location of theaptamer on the glass slide, and thus the protein spotted at thatlocation.

Example of Selex Against Targets

A SELEX procedure as described above was performed utilizing targetmolecules such as amino acids, such as drug compounds, such aslevamisole and cocaine, to produce candidate aptamers, and to yieldaptamer sequences given in the sequence listing above. The sequencesyielded are artificial, non-naturally occurring sequences designedand/or selected for artificially for specific and/or high affinitybinding to drug compounds, such as levamisole and cocaine, theirmetabolites and/or similar/related molecules, where the sequences haveno known natural function, with SEQ IDs 1-190 generally binding to thetarget molecule levamisole, and SEQ IDs 50, 57, 191-290 generallybinding to the target molecule cocaine. Any of the sequences of SEQ IDs1-290 may also have SEQ ID 291 and/or 292 appended to either the 5′-end,3′-end, or both ends, interchangeably, such as to, for example andwithout limitation, provide priming sequences, anchoring sequences (e.g.via hybridization), and/or conformational structure switching behavior(e.g. via switching between hybridized to a nucleic acid andunhybridized while bound to a target molecule), yielding aptamers withthe general structures: 5′-Seq291-SeqX-Seq292-3′,5′-Seq292-SeqX-Seq291-3′, 5′-Seq291-SeqX -3′, 5′-Seq292-SeqX-3′,5′-SeqX-Seq291-3′ and 5′-SeqX-Seq292-3′, where SeqX denotes one of SEQIDs 1-290.

It will be appreciated by those of ordinary skill in the art that thepresent invention can be embodied in other specific forms withoutdeparting from the spirit or essential character hereof. The presentdescription is therefore considered in all respects to be illustrativeand not restrictive. The scope of the present invention is indicated bythe appended claims, and all changes that come within the meaning andrange of equivalents thereof are intended to be embraced therein.

The invention claimed is:
 1. An artificial ligand binding to cocaineconsisting essentially of a non-naturally occurring nucleic acidsequence having the sequence selected from the group consisting of SEQIDs 50, 191-195, and 200-203.
 2. The artificial ligand of claim 1wherein at least one nucleotide is substituted with a non-naturalanalog.
 3. The artificial ligand of claim 1 wherein said non-naturallyoccurring nucleic acid sequence is appended with a 5′-sequence selectedfrom the group consisting of SEQ IDs 291-292.
 4. The artificial ligandof claim 1 wherein said non-naturally occurring nucleic acid sequence isappended with a 3′-sequence selected from the group consisting of SEQIDs 291-292.
 5. The artificial ligand of claim 1 wherein saidnon-naturally occurring nucleic acid sequence is appended with a5′-sequence and a 3′-sequence selected from the group consisting of SEQIDs 291-292.